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WO2015180110A1 - Procédé de préparation d'une microsphère magnétique pour la séparation d'une protéine biologique et utilisation associée - Google Patents

Procédé de préparation d'une microsphère magnétique pour la séparation d'une protéine biologique et utilisation associée Download PDF

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WO2015180110A1
WO2015180110A1 PCT/CN2014/078840 CN2014078840W WO2015180110A1 WO 2015180110 A1 WO2015180110 A1 WO 2015180110A1 CN 2014078840 W CN2014078840 W CN 2014078840W WO 2015180110 A1 WO2015180110 A1 WO 2015180110A1
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magnetic
positive
negative
magnetic microsphere
emulsion
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Chinese (zh)
Inventor
饶微
杜凯
赵莉
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Shenzhen New Industries Biomedical Engineering Co Ltd
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Shenzhen New Industries Biomedical Engineering Co Ltd
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Priority to EP14893409.4A priority Critical patent/EP3150665B1/fr
Priority to PCT/CN2014/078840 priority patent/WO2015180110A1/fr
Priority to US15/126,816 priority patent/US10705079B2/en
Publication of WO2015180110A1 publication Critical patent/WO2015180110A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/20—Esters of polyhydric alcohols or phenols, e.g. 2-hydroxyethyl (meth)acrylate or glycerol mono-(meth)acrylate
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/12—Esters of monohydric alcohols or phenols
    • C08F220/14—Methyl esters, e.g. methyl (meth)acrylate
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L33/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Compositions of derivatives of such polymers
    • C08L33/04—Homopolymers or copolymers of esters
    • C08L33/06—Homopolymers or copolymers of esters of esters containing only carbon, hydrogen and oxygen, which oxygen atoms are present only as part of the carboxyl radical
    • C08L33/10—Homopolymers or copolymers of methacrylic acid esters
    • C08L33/12—Homopolymers or copolymers of methyl methacrylate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
    • C08F220/28—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety
    • C08F220/281—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety and containing only one oxygen, e.g. furfuryl (meth)acrylate or 2-methoxyethyl (meth)acrylate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction

Definitions

  • the present invention relates to the technical field of biological protein separation, and in particular to a method for preparing magnetic microspheres for biological protein separation and an application thereof. Background technique
  • Magnetic nanoparticles (such as Fe 3 () 4 nanoparticles) have properties unique to nanomaterials, such as small particle size, large surface area, high coupling capacity, magnetic responsiveness and superparamagnetism, and can be constant. Aggregation and localization in a magnetic field, absorption of electromagnetic waves in an alternating magnetic field to generate heat, in addition, magnetic nanoparticles can also be modified by surface modification with a variety of active functional groups (such as - OH, - COOH, NH 2, etc.). Therefore, magnetic nanoparticles have broad application prospects in biomedical fields such as biological separation, drug release, and high-heat cancer, and thus have received extensive attention.
  • active functional groups such as - OH, - COOH, NH 2, etc.
  • magnetic microspheres In the field of biology and medicine, magnetic microspheres generally refer to magnetic polymer microspheres, which are magnetic materials developed in recent years, generally using magnetic inorganic particles (such as Fe 3 0 4, etc.) and organic The polymer materials are combined to form a composite microsphere having magnetic properties.
  • the existing magnetic microspheres can achieve a plurality of functional groups on the surface by surface modification or the like, and thus have been widely used in the fields of biology, cytology and separation engineering. Especially in the fields of biological separation and purification, immunoassay, and biological detection, it shows that magnetic microspheres are convenient, fast, and efficient.
  • Magnetic microspheres for biological separation usually need to have the following properties: (1) superparamagnetic; (2) uniform particle size; (3) good dispersibility in aqueous phase; (4) low non-specific adsorption; 5) has surface chemical groups that can be modified. Magnetic microspheres also have significant advantages in protein separation: Magnetic separation technology can be used for large-scale operations; the separation process can be carried out directly in the original sample containing suspended solid particles or additional biological particles; the separation process is simple and rapid.
  • the non-specific uptake of proteins on the surface of magnetic microspheres is a fatal event in the biological separation using magnetic microspheres, which not only reduces the specific separation of microspheres, but also increases the background signal in quarantine assays. Reduce the signal to noise ratio.
  • the strategy to prevent non-specific uptake of proteins by magnetic nanoparticles or magnetic microspheres is to chemically modify their surface.
  • Patent document CN102746529A discloses a method for preparing monodisperse magnetic microspheres by emulsion polymerization, but the magnetic microspheres prepared by the method have non-specific absorption of proteins, have poor performance-to-noise ratio in immunoassay, and have high cost. Not conducive to industrial production.
  • Patent document CN92105584.6 discloses a method for preparing immunomagnetic microspheres by suspension polymerization, in which the surface of ⁇ -F 0 3 or F 0 4 magnetic powder is modified with long-chain fatty acid, and then in styrene. Suspension polymerization was carried out in the solution to obtain immunomagnetic microspheres. Although the magnetic microspheres prepared by the method have low cost, the non-specific adsorption to proteins is also high, and the dispersibility in water is not good.
  • Wang Yu et al. used porous ⁇ -Fe 2 0 3 @ Si0 2 magnetic silica gel as the substrate, first modified with epoxy groups, and then the molecular weight of about 50 ⁇ polymer acid was used to open the epoxy group on the outer surface to form a diol. And then reacting with the residual epoxy groups on the inner surface with octadecylamine, sodium hydrogen sulfite and ethylamine hydrochloride, respectively, and finally obtaining the inner surface of the octadecyl group, the sulfonic acid group and the quaternary ammonium salt group.
  • the outer surface is a new magnetically restricted material based on diol (Wang Yu, a new biological sample pretreatment technology based on functionalization of magnetic microspheres [D], Tianjin University, 2012).
  • the diol group on the outer surface of the material can act as an excretion protein, and the magnetic limiting material having a sulfonic acid group and a quaternary ammonium & base on the inner surface adsorbs the small molecular substance much higher than the inner surface.
  • Octaalkyl material the preparation process of the magnetic globule disclosed in the paper is complicated, and its specific separation effect on biological proteins needs further study. Summary of the invention
  • Another important object of the present invention is to provide a process for the preparation of magnetic microspheres as described above.
  • the inventors of the present invention select a suitable emulsion component and its content based on a large number of studies and experiments, and combine with a magnetic matrix, and the plexus can be reduced without affecting the attachment of a specific protein. Magnetic microspheres that avoid non-specific adsorption with other proteins.
  • the present invention also provides the use of magnetic microspheres as described above for immunodetection.
  • a method for producing magnetic microspheres for biological protein separation comprising the steps of : step a): preparing an emulsion; and step b): dispersing a magnetic magnetic microsphere matrix in the emulsion
  • a dispersion system is obtained;
  • the liquid includes the following components: an acrylic monoester compound, an olefinic acid ester compound, and a trigger.
  • the initiator is preferably a water soluble initiator.
  • the acrylic monoester compound acts as a polymerization monomer
  • the acryl diol ester compound functions as a crosslinking agent.
  • the magnetic microsphere matrix used in the above method is modified to obtain a specific kind of functional group, and under the action of the initiator, the acrylic monoester and the crosslinking agent can be cross-linked and polymerized to coat the magnetic microsphere matrix. Surface, thereby obtaining the magnetic microspheres.
  • the invention adopts the above emulsion to modify the magnetic microsphere matrix not to introduce the active group, and at the same time, the active group is carried by the polymer, but the magnetic microsphere matrix has been activated.
  • the magnetic microsphere matrix is further modified by a polymer layer formed by emulsion polymerization of a specific emulsion, so that the magnetic microspheres can not affect the specific binding of a specific protein (for example, an antigen) through a reactive group. Non-specific absorption with other proteins can be reduced or avoided.
  • a specific protein for example, an antigen
  • Non-specific absorption with other proteins can be reduced or avoided.
  • the magnetic microspheres coated with the polyacrylate polymer prepared by the method provided by the invention are filled with defects such as voids and depressions on the surface (the defects such as voids and depressions are such that they have a certain shape (for example, a Y shape)
  • the interfering antibody is an important cause of non-specific absorption, and the surface is smoother, so the non-specific absorption of other proteins is greatly reduced.
  • the emulsion further comprises a bright ion surfactant and water.
  • the acrylic monoester compound and the acryl diol ester compound are highly hydrophobic, and the magnetic spheroidal matrix has good hydrophilicity, so that the magnetic microsphere and the acrylic monoester compound and the acryl diol ester compound are not sufficiently contacted.
  • a water-soluble surfactant is added, not only is the dispersion of the magnetic microspheres better, but the polymer is easily adsorbed to the surface of the magnetic engraving substrate for polymerization, and by coating the magnetic engraving matrix, It also compensates for some physical defects in the surface of the ball.
  • the emulsion comprises from 0.5 to 30% by weight, based on the total weight of the emulsifier, of an acrylic monoester compound, from 0,05 to 5% by weight of a propylene glycol ester compound, 0.2-2. % by weight of water-soluble initiator, 0, 1 to 1% by weight of water-soluble anionic surfactant, and 62 to 99% by weight of water.
  • the content of the acrylic monoester compound is preferably from 0.1 to 15% by weight, more preferably from 2 to 10%.
  • the content of the W olefinic acid ester compound is preferably 0, 1-2%.
  • the amount of said W acid monoester compound is preferably from 0.5 to 20 wt further 1 1 ⁇ 4, further preferably 2 to 15 wt%.
  • the "acrylic monoester compound” as described above is not limited to a single compound, and may be any mixture of acrylic monoester compounds.
  • the above components such as “acrylic acid diol ester compound” are similar.
  • the specific types and contents of the above respective compounds are not considered separately, and should be considered as a whole, by the synergistic use of the components of the emulsion and their specific contents, the effect of the emulsion on the surface of the modified magnetic sphere is optimized.
  • the content of the propylene glycolate compound is less than 0.05%, the crosslinking polymerization reaction with the monomer is not good, resulting in an unsatisfactory surface modification effect on the magnetic sphere, and finally the invention does not achieve the improvement of the signal-to-noise ratio.
  • the balance of the acryl diol ester compound may interact with the active group S on the magnetic spheroidal matrix, which reduces the ability of the magnetic pheromone to connect with the antigenic protein, which is more unfavorable for detection.
  • the reason for limiting the content of the acrylic monoester compound is as described above.
  • a polymer layer is formed on the surface of the magnetic microsphere matrix, the polymer layer not affecting the magnetic engraving and the specific protein (via the reactive group).
  • the ability to connect, and to reduce the non-specificity of the magnetic microspheres and other proteins, can also ensure the stability of the physical properties of the magnetic microspheres.
  • the crosslinking agent in order to obtain a polymer layer of a suitable thickness and to optimize the polymerization reaction, is synergistic with the monomer and does not bring by-products, thereby better maintaining the magnetic microsphere reactive groups.
  • the white binding ability is the same as the non-specific absorption of the magnetic microspheres and other proteins, and the weight ratio of the acryl diol ester compound is 5-15% by weight of the acrylic monool ester compound, and preferably 10 %.
  • W-olefin monoesters suitable for use in the present invention include, but are not limited to, methacrylic acid esters, ethyl methacrylate, hydroxyethyl methacrylate, n-propyl methacrylate, hydroxypropyl methacrylate, A N-butyl acrylate and hydroxybutyl methacrylate.
  • the acrylate diol compounds suitable for use in the present invention include, but are not limited to, ethylene glycol dimethacrylate, 1,3-propanediol dimethacrylate, 1,3-butanediol dimethacrylate, 1, 4-butanediol dimethacrylate, neopentyl glycol dimethylpropane, and 1,6-hexanediol dimethacrylate.
  • Water-soluble initiators suitable for use in the present invention include, but are not, potassium persulfate, potassium persulfate, and ammonium persulfate; anionic surfactants suitable for use in the present invention include, but are not limited to, sodium dodecyl sulfate, dodecyl Sodium sulfonate, sodium hexadecyl sulfonate and sodium decyl sulfate.
  • the water used is preferably purified water.
  • the non-specific adsorption of the magnetic microspheres and other proteins is reduced without affecting the ability of the magnetic engraving and the later specific proteins to be attached, and does not affect the physical and chemical properties of the magnetic engraving itself, such as magnetic and aqueous phases.
  • a surface-coated polyacrylate polymer layer is finally formed on the surface of the magnetic spheroid substrate. Magnetic microspheres.
  • the magnetic microsphere substrate is subjected to a surface bow "into the reactive group.
  • the reactive group can be bonded to a biological protein.
  • the reactive group is, for example, a thiol group, a hydroxyl group, a carboxyl group, an amino group, an epoxy group, an aldehyde group, an isocyanate group, Cyanate group, cyano group, isocyano group, allyl group, benzyl group, propylene group, maleic group, N-hydroxysuccinimide ester group, carbonyl group, phenolic hydroxyl group, sulfonic acid group, fluorenyl group (including aldoxime, ketone oxime, etc.), azo group, isocyano group, thiol group, and derivatizing groups of all the above functional groups.
  • the magnetic microsphere matrix used in the present invention may be unmodified magnetic according to a known method.
  • the particles are obtained by surface modification, and can also be obtained by direct purchase.
  • the unmodified magnetic particles may be, for example, ⁇ -Fe 2 (3 ⁇ 4, MeFe 2 0 3 (Me di Co, Mn, Ni), Fe 3 0 4 , N Nanoparticles such as i, Co, Fe, Fe-Co and Ni-Fe alloy are preferably ⁇ Fe 2 O 3 and Z or Fe 3 0 4 nanoparticles.
  • Suitable magnetic spheroidal substrates can be, for example, Dynabeads (Dynabe, Norway) ), Estapor microsphere (item Ml-070/60, produced by Merck), Sera-Mag magnetic beads (product number 2415 2.105-050250, manufactured by Thermo Scientific) or Dynabeads magnetic beads (item M-280, manufactured by Life Technologies) .
  • the magnetic microsphere matrix used in the present invention preferably has a particle size of 0.1 to 5 ⁇ 3 : ⁇ .
  • the magnetic microsphere matrix in this particle size range has good dispersibility in the aqueous phase and ensures a large specific surface area.
  • the magnetic microsphere matrix can be solid or hollow, and can also be porous.
  • the shape of the magnetic microsphere matrix is preferably spherical.
  • the spherical magnetic microsphere matrix has a large surface area and the best adsorption effect. In the dispersion system, the concentration of the magnetic microsphere matrix is too large to cause aggregation and precipitation, and if the concentration is too small, the emulsion polymerization effect is poor and the production efficiency is low.
  • the inventors of the present invention have selected the magnetic microsphere matrix concentration in the dispersion system to be 10-400 mg/mL, and preferably 10-200 mg/m:L, and more preferably 10-150 mg after extensive experimentation. /mL, further preferably 20-100 mg/mL. In this concentration range, the magnetic microsphere matrix can be uniformly and stably dispersed in the dispersion system, and a better polymerization effect can be obtained.
  • the polymerization reaction in the step c) of the method is preferably 60. - 90 Torr, more preferably carried out at 70 75 C; it is also preferred to carry out the polymerization for 10 to 40 h, more preferably 15 to 30 h, under stirring.
  • the method of the present invention further comprises the step d) after the step c): solid-liquid separation of the product obtained in the step c), washing the solid to obtain the magnetic spheroid.
  • step a the components of the emulsion are mixed with one another to prepare the emulsion, for example by stirring or sonicating the solid component of the emulsion, and optionally Homogenization treatment (for example, homogenization by high-pressure homogenizer, high-shear emulsification, etc.), the components of the emulsion are sufficiently mixed with each other to obtain an emulsion; in step b), magnetic micro-micro by ultrasonication a ball matrix dispersed in the emulsion; in step d), the solid-liquid separation is performed by magnetic separation or centrifugation; the washing is sequentially washed several times with an organic solvent and water; and the organic solvent is selected from alcohols (for example One or more of an alcohol, ethanol, propanol, isopropanol, decyl alcohol, etc., an ester (such as ethyl acetate, butyl acetate, etc.) and a halogenated hydrocarbon.
  • alcohols for example One or
  • the present invention also provides a magnetic microsphere prepared according to the method as described above, comprising a magnetic microsphere matrix having magnetic properties and a polyacrylate ester coated with the magnetic microsphere matrix.
  • the coating of the polyacrylate material can compensate for certain defects on the surface of the magnetic microsphere substrate, thereby reducing subsequent non-specific adsorption, but does not affect the protein binding ability.
  • the thickness of the surface-coated polyacrylate polymer is particularly important, and if the thickness is too large, the active group a on the surface of the magnetic microsphere substrate is easily covered, thereby affecting its ability to connect with subsequent proteins. If the thickness is too small, the physical modification of the surface of the microsphere substrate is not obtained, so that the ability to reduce non-specific absorption of W is not achieved.
  • the polyacrylic acid ester coated on the surface of the magnetic microspheres prepared by the inventors of the present invention has a thickness of 0.001 to 5 ⁇ M, preferably 5 to 1000 nm, more preferably 10 to 500, and still more preferably 10 to 100. Ling, more preferably 50mii - 100iim.
  • the polyacrylate polymer layer in the thickness range is favorable for not affecting the connection between the surface group of the late magnetic microsphere and the specific protein, reducing or avoiding the non-specific adsorption of the magnetic microsphere and other proteins, and avoiding affecting the magnetic micro
  • the present invention also provides the use of magnetic microspheres prepared according to the method described above for biochemical detection, for example, in the detection of rabbit disease, especially in the quantitative analysis of chemiluminescence immunoassays.
  • biochemical detection for example, in the detection of rabbit disease, especially in the quantitative analysis of chemiluminescence immunoassays.
  • Such applications for example Chemiluminescent immunodetection of various specific total antibodies, specific IgG, IgM antibodies in human serum.
  • the magnetic microspheres provided by the present invention can be used against Toxoplasma gondii IgG antibody, Toxoplasma IgM antibody, glutamate decarboxylase antibody, rubella virus IgG antibody, rubella virus IgM antibody, cytomegalovirus IgG antibody, Chemiluminescence immunoassay for detection of at least one of cytomegalovirus IgM antibody, type I/II type simple cell virus IgG antibody, type simple cell virus IgM antibody, Epstein-Barr virus IgG antibody, and Epstein-Barr virus IgM antibody.
  • the method for preparing the magnetic microspheres prepares the magnetic microsphere matrix by preparing and using a suitable emulsion, and modifying the surface of the magnetic microsphere substrate by emulsion polymerization, thereby obtaining a bio-separation.
  • Magnetic microspheres When used for the separation of ff ⁇ from biological proteins, the magnetic microspheres significantly reduce the non-specific risk of subsequent proteins without affecting the ability to bind to specific proteins, and solve a major problem in the separation of biological proteins. The problem provides a new option for separation engineering that achieves high protein-specific risk.
  • the magnetic microspheres obtained by this method have good dispersibility, are suitable for large-scale operations, and the separation process is simple and easy.
  • the preparation method has simple operation steps, and the raw materials used are cheap and easy to obtain, so that it has great utilization value and economic significance. detailed description
  • the magnetic microsphere matrix with a carboxyl functional group used in the present example was prepared in accordance with the examples in Chinese Patent No. CN92105584.
  • composition of the emulsion and its mass percentage are as follows:
  • the magnetic microsphere matrix obtained in the step 1) was dispersed in the emulsion prepared in the step 2) at a concentration of 20 mg/mL, and the magnetic microsphere matrix was uniformly dispersed by ultrasonic treatment to obtain a dispersion system.
  • the dispersion obtained in the step 3) was stirred and reacted at 75 ° C for 30 hours, and the supernatant was separated by a magnet, and the solid was washed 3 times with the alcohol and washed 5 times with water to obtain magnetic microspheres.
  • Test sample selection Toxoplasma gondii] gG (TOXCMgG) negative and positive samples in 0 cases, glutamate decarboxylase (GAD65) negative and positive samples in 10 cases, and cytomegalovirus: IgM (CMV IgM) negative negative and There were 10 cases of ffl samples. All of the above samples were confirmed by clinical validation.
  • Antigen magnetic encapsulation The magnetic microspheres prepared in this example were coated with TOXO antigen GAD65 antigen and CMV antigen, and diluted to a working concentration of 0.5 mg/niLo in phosphate buffer PBS.
  • luminescent label ⁇ ABEI (N-(4-aminobutyl)-N-ethylisoluminol) labeled mouse anti-human monoclonal IgG antibody (anti-hlgG-ABEI) (diluted with PBS) Up to 0.02 g/mL), ABEI-labeled Staphylococcal Protein A (SPA-ABEI) (diluted to 0.04 iig/niL with PBS), ABEI-labeled murine anti-human monoclonal IgM antibody (anti-hlgM-ABEI) (diluted to PBS to 0.02 ig/mL) luminescent markers detected as TOXO IgG, GAD65 and CMV M, respectively.
  • ABEI N-(4-aminobutyl)-N-ethylisoluminol
  • SPA-ABEI ABEI-labeled Staphylococcal Protein A
  • SPA-ABEI diluted to 0.04
  • Test sample selection Normal human serum, clinically confirmed TOXO IgG, GAD65 and CMV IgM negative.
  • luminescent markers ⁇ ABEI (N-(4-aminobutyl)-Nethylisoluminol) labeled mouse anti-human monoclonal IgG antibody (anti-hlgG-ABEI) (with PBS at 1) : 4000 for dilution), ABEI-labeled staphylococcal protein A (SPA-ABEI) (diluted 1:3000 with PBS), ABEI-labeled murine anti-human monoclonal IgM antibody (anti-hlgM-ABEI) (with PBS at 1: 4000 was diluted) as luminescent markers for TOXO IgG, GAD65 and CMV IgM, respectively.
  • SPA-ABEI staphylococcal protein A
  • ABEI-labeled murine anti-human monoclonal IgM antibody anti-hlgM-ABEI
  • Antigen magnetically coated microspheres coated with the sputum antigen GAD65 antigen and CMV antigen Magnetic microspheres were diluted to a working concentration of 0.5 mg/mL with phosphate buffered PBS.
  • a magnetic microsphere matrix was prepared according to the method disclosed in Chinese Patent No. CN92105584, and its surface active group connection property and non-specific absorption property were measured. The specific measurement conditions and procedures were the same as those in Step 5) and Step 6) in Example 1, except that the magnetic microsphere matrix prepared in the present comparative example was used in place of the magnetic properties in Steps 5) and 6) of Example 1. Microspheres. The results are shown in Table 1-4.
  • the TQXO antigen, the GAD65 antigen and the CMV antigen coated with the magnetic microspheres prepared in Example 1 of the present invention produced good binding to the antibodies in the corresponding antibody-positive samples, respectively, and were negative for the corresponding antibodies.
  • the sample can detect a completely negative signal. This indicates that the surface active groups of the magnetic microspheres prepared in Example 1 have excellent coupling ability with proteins, ie, antigens, and the polyacrylic acid ester polymer on the surface of the magnetic microspheres does not affect the active groups on the surface of the magnetic microspheres at all. Protein binding.
  • Example 2 the non-specific absorption properties of the magnetic microspheres prepared in Example 1 of the present invention and the antibodies in the sample were significantly lower than those of the magnetic microsphere matrix modified in the emulsion prepared in the present invention without the use of the emulsion prepared in the present invention. Suction performance, and, for some special samples, the test results compared to 1 more appear to be inconsistent with the clinical sample. The above experiments and data show that the magnetic microspheres of Example 1 of the present invention have a remarkable effect of reducing or avoiding adsorption of non-specific proteins.
  • Example 2 the magnetic microspheres of Example 1 of the present invention have a remarkable effect of reducing or avoiding adsorption of non-specific proteins.
  • the magnetic microsphere substrate of the present embodiment was prepared in accordance with Examples 1-6 of Chinese Patent Publication No. CN102746529A. That is, first, polystyrene polymer seed particles are prepared; then the seed particles are modified using an emulsion containing polyvinylpyrrolidone, divinylbenzene, styrene, and toluene (porogen), and emulsion polymerization is performed to obtain porosity. Polystyrene particles; the porous polystyrene particles are nitrated using nitric acid; and then FeSCX ⁇ iron is used to bind to the porous polystyrene particles; then the obtained particles are coated and carboxyl functionalized, and the present embodiment is obtained. For example, a magnetic spheroidal matrix with a carboxyl functional group is used.
  • composition of the emulsion and its mass percentage are as follows - hydroxyethyl methacrylate 0.5%;
  • the magnetic microsphere matrix obtained in the step 1) was dispersed in the emulsion prepared in the step 2) at a concentration of 40 mg/mL, and the magnetic microsphere matrix was uniformly dispersed by ultrasonic treatment to obtain a dispersion system.
  • the dispersion obtained in the step 3) was stirred and reacted at 80 ° C for 8 hours, and the supernatant was separated by a magnet, and the solid was washed 3 times with the alcohol and washed 5 times to obtain magnetic microspheres.
  • Example 4 The test was carried out in accordance with step 5) of Example 1, except that the magnetic microspheres used were replaced by the actual ones.
  • Example 4) The obtained magnetic microspheres were prepared. The results are shown in Tables 5 to 7.
  • Example 2 The experiment was carried out in accordance with the procedure of Example 2, except that the emulsion in Example 2 was replaced with the following emulsion:
  • composition of the emulsion and its mass percentage are as follows:
  • the magnetic microsphere matrix was prepared in accordance with Examples 1-4 of Chinese Patent Publication No. CN 102746529 A, and the surface active group bonding property and non-specific adsorption property were measured.
  • the specific measurement conditions and procedures are the same as those in the step 5) and the step 6) in the embodiment 2, except that the magnetic microsphere substrate prepared in the present comparative example is used instead of the embodiment 2, step 5) and the step
  • the magnetic micro-sample of the magnetic micro-comparative example 2 of Example 3 was negative.
  • the illuminating signal intensity of the spherical sample The luminescent signal of the spherical matrix Test item Sex / Yang Luminous signal intensity and yin
  • the magnetic micro-sample of the magnetic micro-comparative example 2 of Example 3 was negative.
  • the illuminating signal intensity of the ball sample The luminescent signal of the ball body Test item Sex / Yang Luminous signal intensity and yin
  • the TOXO antigen, the GAD65 antigen and the CMV antigen coated with the magnetic microspheres prepared in Examples 2 and 3 of the present invention respectively produced good binding with the antibody in the corresponding antibody-positive sample, and for the corresponding antibody.
  • a negative sample can detect a 3 ⁇ 4 completely negative signal. This indicates that the surface active groups of the magnetic microspheres prepared in Examples 2 and 3 have excellent coupling ability with proteins, ie, antigens, and the polyacrylate polymer on the surface of the magnetic microspheres does not affect the active groups on the surface of the magnetic microspheres at all. Protein binding.
  • the magnetic microsphere substrate of the present embodiment was prepared in accordance with Examples 1-6 of the Chinese Patent Publication No. CN102746529.
  • composition of the emulsion and its mass percentage are as follows:
  • the magnetic microsphere matrix obtained in the step 1) was dispersed in the emulsion prepared in the step 2) at a concentration of 50 mg/mL, and the magnetic microsphere matrix was uniformly dispersed by ultrasonic treatment to obtain a dispersion system.
  • the dispersion obtained in the step 3) was stirred and reacted at 75 ° C for 28 hours, and the supernatant was separated by a magnet.
  • the solid was washed three times with methanol and washed with water five times to obtain a magnetic spheroid. 5) Detection of the connectivity of surface active groups of magnetic microspheres
  • Example 2 The test was carried out in accordance with the procedure 5) of Example 1, except that the magnetic microspheres used were replaced with the magnetic microspheres prepared in the step 4) of the example.
  • the binding ability of the active group of the magnetic microsphere prepared in this example to the antigen protein is similar to that of Examples 1 and 2, and the antigen of the magnetic microsphere is coated with The antibody in the sample binds very well, while the negative sample shows a completely negative signal.
  • Example 6 of Example 1 The test was carried out in accordance with the procedure of Example 6 of Example 1 except that the magnetic microspheres used were replaced with the magnetic spheroids prepared in the step 4) of the present example.
  • the magnetic microspheres prepared in this example coated with the antigen are similar to the examples 1 and 2 in the adsorption detection of the antibodies in the sample, and the non-specific adsorption is very less.
  • Example 5
  • the magnetic microsphere substrate used in the present example was prepared in accordance with Examples 1 - 4 of Chinese Patent Publication No. CN 102746529 A.
  • composition of the emulsion and its mass percentage are as follows:
  • the above components were mixed in proportion, sonicated, and the solid components therein were all dissolved, and then homogenized in a high pressure homogenizer for 20 minutes.
  • the magnetic microsphere matrix obtained in the step 1) was dispersed in the emulsion prepared in the step 2) at a concentration of 50 mg/mL, and the magnetic globule matrix was uniformly dispersed by ultrasonic treatment to obtain a dispersion system.
  • the dispersion obtained in the step 3) was stirred at 70 ° C for 24 hours, and the supernatant was separated by a magnet. The solid was washed 3 times with an alcohol and washed 5 times to obtain magnetic microspheres.
  • the binding ability of the active group of the magnetic microsphere prepared in this example to the antigen protein is similar to that of Examples 1 and 2, and the antigen of the magnetic microsphere is coated with The antibody in the sample binds very well, while the negative sample shows a completely negative signal.
  • test was carried out in accordance with the procedure 6) of the repeated example I, except that the magnetic microspheres used were replaced with the magnetic microspheres prepared in the step 4) of the present example.
  • the magnetic microsphere substrate of the present embodiment was prepared in accordance with Examples 1-4 of Chinese Patent Publication No. CN102746529A.
  • composition of the emulsion and its mass percentage are as follows - hydroxypropyl methacrylate 12%;
  • the magnetic microsphere matrix obtained in the step 1) was dispersed in the emulsion prepared in the step 2) at a concentration of 100 mg/niL, and the magnetic microsphere matrix was uniformly dispersed by ultrasonic treatment to obtain a dispersion system.
  • the dispersion obtained in the step 3) was stirred and reacted at 70 ° C for 20 hours, and the supernatant was separated by a magnet.
  • the solid was washed 3 times with ethanol and washed 5 times with water to obtain magnetic microspheres.
  • Example 4 The test was carried out in accordance with step 5) of Example 1, except that the magnetic microspheres used were replaced by the actual ones.
  • the binding ability of the active group of the magnetic microsphere prepared in this example to the antigen protein is similar to that of Examples 1 and 2, and the antigen of the magnetic microsphere is coated with The antibody in the sample binds very well and exhibits a 3 ⁇ 4 completely negative signal for the clear sample.
  • Example 6 of Example 1 The test was carried out in accordance with the procedure of Example 6 of Example 1 except that the magnetic microspheres used were replaced with the magnetic microspheres prepared in the above step 4). From the experimental result data (not listed here), the magnetic microspheres prepared in this example coated with the antigen are similar to the examples 1 and 2 in the adsorption detection of the antibodies in the sample, and the non-specific adsorption is very less. Modifications within the spirit and scope of the invention will be apparent to those skilled in the art. Further, it should be understood that various aspects of the invention, various parts of the various embodiments, and various features listed herein may be combined or in whole or in part.

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Abstract

L'invention concerne un procédé de préparation d'une microsphère magnétique pour la séparation de protéines biologiques. Une matrice de microsphère magnétique est traitée par la formulation et l'utilisation d'un liquide émulsifié approprié, et la modification de la surface de la matrice de microsphère magnétique est réalisée par polymérisation par émulsion, ce qui permet d'obtenir une microsphère magnétique revêtue d'une couche de polymère de polyacrylate. Ledit liquide émulsifié comprend, en son sein, les constituants suivants : des composés monoester d'acide acyclique, des composés glycol d'acide acyclique, des initiateurs et éventuellement des agents tensioactifs anioniques et de l'eau. La microsphère magnétique réduit de manière significative l'adsorption non spécifique d'autres protéines, sans affecter la capacité de liaison à une protéine spécifique, lorsqu'elle est utilisée dans la séparation de protéines biologiques. Une nouvelle sélection est prévue pour réaliser la technique de séparation par adsorption de protéine de spécificité élevée.
PCT/CN2014/078840 2014-05-29 2014-05-29 Procédé de préparation d'une microsphère magnétique pour la séparation d'une protéine biologique et utilisation associée Ceased WO2015180110A1 (fr)

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PCT/CN2014/078840 WO2015180110A1 (fr) 2014-05-29 2014-05-29 Procédé de préparation d'une microsphère magnétique pour la séparation d'une protéine biologique et utilisation associée
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CN120479321A (zh) * 2025-07-14 2025-08-15 杭州博岳生物技术有限公司 一种自抗污超亲水免疫磁性微球及其应用

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